BioHack AcademyBioreactor Design

• Antibiotics• Steroids / hormones• Vitamins• Proteins• Sugars• Acids

Bioreactor value pyramid

Pharma

Food

Fibers

Fuel

HeatVolume

Value

Bacterial growth curve - Batch

LagPhase

LogPhase

Declining GrowthPhase

Endogenous RespirationPhase

Food curve

Bact

eria

l Mas

s /

Amou

nt o

f Foo

d

Time

Growth Curve

Primary products

LagPhase

LogPhase

Declining GrowthPhase

Endogenous RespirationPhase

Food curve

Bact

eria

l Mas

s /

Amou

nt o

f Foo

d

Time

Growth Curve

Extend log growth phase

Secondary products

LagPhase

LogPhase

Declining GrowthPhase

Endogenous RespirationPhase

Food curve

Bact

eria

l Mas

s /

Amou

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f Foo

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Time

Growth Curve

OpenStax College CC-BY-SA 3.0

Intra vs extra cellular

Growth strategies

Batch Fed Batch Chemostat

• Simple design• Many cells per volume• Uniform distribution• Simple oxygen supply• Low energy use

Bioreactor Wishlist

• Stirrer tank• Air-lift• Membrane• Immobilized cells• Cell culture• Solid state• Photobioreactor• Microbioreactors• Animals

Bioreactor Typeshttp://nptel.ac.in/courses/102103016/module4/lec36/3.html

Bioreactor hackshttp://www.instructables.com/id/An-Algae-Bioreactor-from-Recycled-Water-Bottles/ http://www.instructables.com/id/Biogas-at-home-Cheap-and-Easy/

Briefcase Biotec https://www.youtube.com/watch?v=yDarDR4vi1A

Membrane reactor: Fuel Cells

PublicDomain

Soil based fuel cellsMFCGuy2010 - CC-BY-SA 3.0

Waag Society - CC-BY-SA 3.0

Bio mud batteryhttp://www.instructables.com/id/Create-your-own-mud-battery-Bouw-je-eigen-modderba/

Waag Society - CC-BY-SA 3.0

BIOMONSTAAARhttp://biomonstaaar.com

Flu vaccine production in eggs

Insect cell line

Sugar cane to ethanol

Second generation

Geometry of standard stirred tank

Brazilian Journal of Chemical Engineering

for aerobic reactions

Example designViolacein production

My search for J. lividum

• „Janthinobacterium lividum” +• „growth conditions”• „violacein pathway”• „violacein genes”• „patent”• „yield”• „inhibition”• „extraction”

http://www.housatonicrods.com/rivrpics_2007.html

Violacein pricing

Janthinobacterium lividum

Wikipedia tells me:• Gram negative• Aerobic

Violacein production from glycerol

Waag Society - CC-BY-SA 3.0

Production pathway?

P. Roqueforti eating lactate

C3H5O3- + 3O2 + H+ -> 3CO2 + 3H2O

Acid is consumed

Violacein genesHornung et al. - The Janthinobacterium sp. HH01 Genome Encodes a Homologue of the V. cholerae CqsA and L. pneumophila LqsA Autoinducer Synthases (2013)

Production pathway?

Antonio, R.V. and Creczynski-Pasa, T.B. (2004) Genetics analysis of violacein biosynthesis by C. violaceum. Genet Mol Res 3, 85–91.

Tryptophan

Other interesting things:

• J. lividum produces a metallo-β-lactamase conferring resistance to

several β-lactam antibiotics

• Violacein:

• C20–H13–N3–O3

• molecular weight of 343·33

• insoluble in water

• soluble in alcohols as methanol, ethanol and acetone

• maximal absorption in a solution of methanol is at 585 nm

Rossolini, G.M., Condemi, M.A., Pantanella, F., Docquier, J.D., Amicosante, G. and Thaller, M.C. (2001) Metallo-β-lactamase producers in environmental microbiota: new molecular class B enzyme in Janthinobacterium lividum. Antimicrob Agents Chemother 45, 837–844.

Blosser, R.S. and Gray, K.M. (2000) Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassey for N-acyl homoserine lactone autoinducers. J Microbiol Methods 40, 47–55.

Production inhibition

Pantanella, F., Berlutti, F., Passariello, C., Sarli, S., Morea, C. and Schippa, S. (2007), Violacein and biofilm production in Janthinobacterium lividum. Journal of Applied Microbiology, 102: 992–999. doi: 10.1111/j.1365-2672.2006.03155.x

Production conditions

Donaldson - Treating Seawater Biofilms with Violacein Extracts (2013)

Patent - USPTO

EXAMPLE 1 PRODUCTION AND EXTRACTION OF THE BIOACTIVE PIGMENT FROM THE CULTURE OF CHROMOBACTERIUM SP. NIIST-CKK-01 A loopful of 24 hrs old pure culture Chromobacterium sp. NIIST-CKK-01 from solid agar medium (LB agar or Nutrient agar) was inoculated with 50 ml of the growth medium (0.5% Yeast extract and 1.5% Peptone) taken in a 250 ml Erlenmeyer flask. Alternatively, 10% (v/v) of 24 hour old pure culture of Chromobacterium sp. NIIST- CKK-01 in LB broth was also used as inoculum. The pH of the medium was 7. The flasks inoculated with Chromobacterium sp. NIIST-CKK-01 were subsequently incubated in a rotary shaker at ambient temperature (30 °C) and 200 rpm for 24 hours. The deep purple purple-blue pigment starts appearing in the medium by about 6 hours of incubation and continued beyond biomass increase (Fig 1). After 24 hrs of incubation, the bacterial biomass with pigment was centrifuged at 9676.8 x g and 4 °C for 10 minutes. After centrifugation, the clear supernatant was removed. The pellet containing biomass and pigment was mixed thoroughly with 5 ml of extra pure methanol. The mixture was centrifuged again at 9676.8 χ g and 4 °C for 10 minutes to separate the cell pellet from the solvent-pigment mixture. The pigment extraction was repeated twice using fresh solvent as described. All the pigment extracted solvent pooled together and the pigment was concentrated by normal vacuum drying in a desiccator. The quantity of biomass and pigment produced could be accounted by measuring optical density at 600 nm and 575 nm respectively. The yield of pigment by this method was about 1.0 g pigment/g of dry biomass in 24 hrs. HPLC analysis is carried out for checking the purity of the pigment produced using an ODS column (Lichrospher-100; Merck) with acetonitrile (40%) at lml/min as mobile phase and using UV-VIS detector at 575 nm (Figure 2). UV-VIS absorption spectra indicated maximum absorption at 575 nm, typical of violacein and its derivatives (Figure 3). EXAMPLE 2

Process for the production of violacein and its derivative deoxyviolacein containing bioactive pigment from Chromobacterium sp. (MTCC5522)

J. Lividum canvas

material

BioFactorycanvas

species

volume

light

oxigen

temp.

absorbance

pH

mass

stirrer

time

O2

observationsday #

day #

day #

day #

day #input

C

N

P

O2

S

Yes

15

48h

330 ml

No

NutrientBroth

GlycerolTryptoph.

No1 mL?

?

585

Genetic construct for E. coli

Fang et al. Microbial Cell Factories 2015 14:8   doi:10.1186/s12934-015-0192-x

Synbiota - ScienceHack

Twitter @synbiota Twitter @GentleDNA

These slides are published by Waag Society under CC-BY-SA 4.0 license